Apparatus for production and extraction of charged particles

ABSTRACT

The apparatus comprises a sample, an optical element ( 4 ) in the form of a truncated pyramid having at least one reflective surface ( 1   a ) and a hole ( 7 ). A laser ( 18 ) directs radiation on to the sample via the reflective surface ( 1   a ) and the charged particles are extracted and directed along an extraction axis through the hole ( 7 ).

FIELD OF THE INVENTION

This invention relates to apparatus for the production and extraction ofcharged particles, particularly such apparatus wherein the emission ofcharged particles is stimulated by light irradiation.

BACKGROUND OF THE INVENTION

The emission of charged particles stimulated by light irradiation is afundamental physical process used in many modern analytical techniques.One of the main requirements of a system using such a process is tocombine the most efficient generation and transmission to the desireddestination of the emitted charged particles with the most effective useof the light irradiation.

Light Stimulated Charged Particle Emission.

A fundamental physical process first noted when electrons were seen tobe emitted when irradiated by a light source, light stimulated particleemission forms the basis of many materials analysis techniques. Nowadaysthe full range of the electromagnetic spectrum may be utilised and thecharged particle may be an electron, ,positron, anion or cation.

Charged Particle Extraction.

For the most efficient extraction of charged particles in an electricfield, it is generally accepted that the closer the initial trajectoriesare to being parallel with the axis of extraction, the higher theefficiency. In this case the efficiency refers to the number of chargedparticles that are transferred from the region of their emission totheir destination with the desired parameters optimised. For example,the parameter may be energy or time dispersion and the optimisation isto minimise these parameters. One solution to optimising theseparameters is to exclude those that do not satisfy the requirements.This is often achieved by physically preventing them being detected.This reduces the number of charged particles analysed and sensitivityproblems may arise if the source is of low emissivity. A compromise mustthen be made between sufficient sensitivity and the optimisation of therequired parameters to make the analysis meaningful.

An important factor in the efficient extraction of charged particles isthe coincidence or near coincidence of the axes of emission andextraction.

MALDI Analysis

Presently such a system is widely, although not exclusively, used in theMatrix Assisted Laser Desorption and Ionisation (MALDI) technique usedfor the analysis of biological, biochemical and polymeric materials asdescribed in Protein & Polymer Analyses up to M/Z 100,000 by LaserIonisation Time-of-Flight Mass Spectrometry, K. Tanaka et al. RapidComm. Mass Spectrom. Vol.2, pp 151-153, 1988. Key to this technique arethe desorption and ionisation processes which allow intact largemolecules to be extracted from the sample, a so-called “soft ionisation”technique. Typically, a substance called the matrix, which is insolution, is combined with the substance to be analysed, the analyte,also in solution, on a sample stub or slide. This combination is allowedto dry and then placed inside an evacuated chamber. Emission andionisation of intact large molecules is then stimulated by the use of alaser. The ionised molecules produced are then accelerated away from thesample by an electric field and into an analyser. The use of a pulsedlaser allows a relatively simple and low cost Time-of-Flight (ToF) massanalyser to be used for obtaining information from the sample, e.g.identifying the molecular weight.

In MALDI, the matrix is chosen for its good absorption of energy fromthe laser and, additionally, through a photon and/or chemical ionisationprocess, provides a mechanism that produces a quantity of ions from theanalyte for analysis. To obtain the best results from the wide range ofanalytes which can be analysed by the MALDI technique many differentmatrices are used, each offering some different characteristics whichare dependent on the chemistry of the analyte. Therefore, it isnecessary to match the matrix to the analyte to be analysed. However,this condition of matching the matrix and analyte to achieve the bestlevel of information often gives rise to non ideal conditions foroptimised ion extraction. These non ideal conditions manifest themselvesin the form of surface roughness and inhomogeneities in the combinationof matrix and analyte. In the case of poor mixing of matrix and analyte,care in the preparation of the sample can alleviate many of theproblems, as described, for example, in “Growing Protein-doped sinapinicacid crystals for Laser Desorption,” Xiang and Beavis, Organic MassSpectrometry, Vol.28, pp 1424-29, 1993. However, surface roughness canbe very difficult to eliminate, as the drying process often leads tounavoidable crystallisation. This crystallisation can give rise tosurface roughness of the order of 10 to 50 microns or more as describedin “A comparison of matrix/analyte protein surface distributions inMALDI samples by XPS analysis ” by A Smith et al. Proceedings of the45th ASMS Conference, p1041, 1997.

Another issue with biological and biochemical analysis is efficientsample utilisation. In many cases the amount of a substance that isavailable for analysis is very limited so effective ionisation andextraction is often an important factor in MALDI analysis. Model of theMALDI Process

The fundamental MALDI process of ejection and ionisation is notcurrently well understood. In part this is because of the large varietyof matrix/analyte combinations. That is to say, what might be understoodfor one particular matrix/analyte combination may not apply to adifferent combination. This makes a common model difficult, if notimpossible, to define. Another difficulty in performing an analysis ofboth the physical and chemical processes is that they take place in theorder of tens of nanoseconds making measurement extremely difficult.

It is however possible to have a good qualitative model of the MALDIprocess. In this model the pulsed laser irradiates a region of thesample. Some molecules in this region receive sufficient energy toescape from the sample. This is called laser ablation. The laser ispulsed at a high energy, but with a short duration, of the order ofnanoseconds, to remove some of the material from the sample. Thismaterial is ejected in a supersonic plume away from the surface of thesample. Either during or shortly after ablation, a number of the samplemolecules become ionised. They can then be extracted by the applicationof a suitable electric field.

Angular Distribution of Ablated Material.

FIG. 1 shows where the pulsed laser beam 1 irradiates an area of thesample 2 which is ablated 3. FIG. 1 also shows the angular distribution6 for a point source which has the largest number of particles ablatedalong trajectories perpendicular to the surface. Extrapolating thispoint distribution across the surface gives rise to the distributionshown by the dotted line 7. In general, the distribution of ablatedmaterial has an axis of ejection 5 perpendicular to the surface and onlyhas a weak dependence on the angle of incidence 4 of the irradiatinglight source 1. This is for the case of a perfectly flat surface.However, if we compare this to the case of a rough or curved surfacethen the angular distribution of the ablated material is modified. InFIG. 2 we see a curved surface which we can treat as a series ofstraight line segments or tangents 2 joined together as shown which isirradiated by a light source 4. By treating each of these tangents as alocal surface we then can define a local axis 5 which is perpendicularto that local surface 2. Each local surface 2 therefore has its ownlocal axis 5. The distribution of ablated material 3 from the localsurface about the local axis 5 is the same as for a point source asshown in FIG. 1. If we now extrapolate these to obtain the distribution4 over the whole curved surface that is irradiated we see that it isconsiderably broadened compared to the case of the flat surface. By alogical extension of the argument this broadening of the angulardistribution can also be seen to be true for a concave as well as theillustrated convex surface.

This broadening of the angular distribution arises wherever the localaxis of emission varies across the sample surface, i.e. as in the caseof a rough surface. Referring back to the earlier comments on chargedparticle extraction, any increase in the angular distribution of chargedparticles leads to reduced efficiency and it is therefore desirable tominimise this angular distribution. This is in general agreement withthe work by Vorm et al entitled “Improved Resolution and Very HighSensitivity in MALDI ToF of Matrix Surfaces made by Fast Evaporation.”Anal.Chem.Vol.66, No.19, pp 3281-3287, 1994.

Shadowing.

In FIG. 3a we see the situation where the axis of irradiation 1 is at anangle with respect to the axis of extraction 9. This can give rise toshadowing of an area 3 of the sample surface 4 from the lightirradiation 5. This is due to the surface structure 6. Hence it can beseen that not all of the sample in the area of interest 2 is irradiated.Also, as can be seen in FIG. 3b, charged particles are emitted withlocal axes of emission 7 which have large angular deviations relative tothe axis of extraction 9.

FIG. 4a shows the case of near perpendicular irradiation of the sample4. In this situation the axis of irradiation 1 and the axis ofextraction 9 are closer to being coincident than is the case in FIG. 3a.It can be seen that the irradiated area 2α and 2β is larger than theirradiated area 2 in FIG. 3a and the shadowed area 3 caused by thesurface structure 6 is smaller when compared to the same region 3 inFIG. 3a. In FIG. 4b it can also be seen that the number of ions that areemitted with local axes of emission 7 nearly coincident with the axis ofextraction 9 is increased.

Ablation Volume.

The amount of material ablated is dependent on the amount of energydeposited and effectively absorbed in a volume of the sample near to thesurface. As shown in FIGS. 3b and 4 b, the shaded area 8 indicates thevolume of the sample that absorbs the energy from the irradiating light.In both cases the irradiated volume is approximately the same. However,the volume beneath surfaces perpendicular to the axis of extraction isgreater in the case of near perpendicular irradiation. This demonstratesthat the emission of material from the surfaces that have the smallestangular deviation from the axis of extraction is enhanced. Acorresponding decrease in emission is seen in areas that have largeangular deviations from the axis of extraction.

Optimisation of Efficiency.

From the argument presented it can be seen that it is highlyadvantageous for the axis of irradiation to be as closely coincidentwith the axis of extraction as is practical. This minimises emissionfrom the areas of the sample that reduce the efficiency of the chargedparticle extraction, i.e. surfaces that are not perpendicular to theaxis of extraction, and maximises emissions from surfaces that are,thereby improving the efficiency of the ion extraction.

Also, sample preparation becomes less critical as it allows the user tofocus on obtaining the best results from the chemistry without having tobe overly concerned with the additional problem of attaining a suitablyflat sample.

A further benefit is an increase in the sample utilisation allowingsmaller sample quantities to be used. This is highly-desirable in thecase of many biological and biochemical samples where often onlypicomoles or femtomoles of material is available for the analysis.

Prior Art

In general, the prior art has failed to satisfy the requirements for themost efficient charged-particle generation and transmission of theemitted charged particles to the desired destination combined with themost effective use of the light irradiation. In the majority of cases,compromise has been made in the light irradiation which is typicallyincident on the sample at an angle in the range from 45 degrees to 60degrees with respect to the axis of extraction. This compromise ispreferred since introducing asymmetry into the extraction system reducesthe overall efficiency to a far greater degree.

FIG. 5 shows a typical system as used in MALDI. The system comprises alaser 2, a neutral density filter 3, a mirror 4, a focusing lens 1, awindow 5, an evacuated chamber 6, a sample 7, a grid 9 and anelectrostatic lens 8. In a typical system, the source of lightirradiation is a pulsed laser 2, its power being attenuated by theneutral density filter 3 to the desired level. The laser beam 11 is thenreflected by the mirror 4 towards the lens 1 where it is focused to passthrough the window 5 to irradiate the sample 7. Charged particlesemitted are then accelerated along the axis of extraction 12 by theapplication of an electric field. An extraction system comprising thesample 7, the grid 9 and the focusing lens 8 is used to accelerate andfocus the emitted charged particles (indicated by the trajectoryenvelope 10) along the axis of extraction 12. As can be seen, thetypical system does not address the problem of efficient extraction asdiscussed previously. Other methods of introducing light irradiation toa sample in an electrostatic field are now discussed and their meritsand demerits are presented.

One method of irradiating the sample involves using a fibre optic guideto direct the irradiating light to the sample. This is described in U.S.Pat. No. 5,118,937. FIG. 6 shows such a system wherein a laser beam 7 isfocused by a lens 5 into an optical guide 3 and focused onto the sample2 by a lens 6 at the other end of the guide. Ions 4 generated in thismanner are extracted through a grid 1 along the extraction axis 8. Thissuffers similar demerits as described with reference to FIG. 5, with theadditional drawback that the electrically insulating material of theoptical guide is situated inside the electric field region; this canlead to an asymmetric extraction field due to an accumulation of staticelectrical charge on the optical guide.

FIG. 7 shows a further example of the prior art where a sampleobservation and illumination system is implemented in addition to thelight irradiation. In this case, the sample observation is at an anglesimilar to that used for the light irradiation, but viewing is from adifferent axis. This is the simplest implementation of a sampleobservation system. A laser beam 1 irradiates the sample 4 after beingfocused by the lens 2. The sample is illuminated with visible light 6and the sample is observed by forming an image at the viewing position 5using the lens 7. Ions 10 are extracted along the axis of extraction 9by an electric field through an extraction grid 3 and focused by anelectrostatic lens 8. The disadvantages of this system are the reducedefficiency of the extraction system and the poor correlation betweenobservation and the irradiation point. Also because the angle ofobservation is acute, a large depth of field is required to obtain auseful field of view.

Systems where the observation axis and the irradiation axis arecoincident are achievable by the use of Dichroic mirrors, but suchsystems are complex, expensive and inflexible. Therefore, these systemswill not be considered any further.

A method for the perpendicular or near perpendicular irradiation of asample is shown in FIG. 8. Here an angled mirror 4 having a hole 7 isplaced so that the hole lies on the axis of extraction 8. In the case ofa mirror inclined at 45 degrees, the axis of irradiation 9 is usuallyperpendicular to the axis of extraction 8. The irradiating light 5 isfocused by a lens 1 and is reflected by the mirror 4 towards the sample3. Ions 11 generated by the irradiating light 5 are accelerated alongthe axis of extraction 8 by an electric field between the sample and thegrid 2 and pass through the hole 7 in the mirror 4 via the electrostaticlens 10. In this system, the mirror 4 is asymmetric with respect to theextraction axis. In general, if the best extraction efficiency is to beachieved, then asymmetry of the extraction system should be avoided.This typically requires the mirror to be in a region free from electricfields. This can place serious constraints on the design of theextraction optics. Furthermore, any irradiating light 6 which is notreflected, but passes through the hole 7 is lost and hence the powerfrom the irradiating source is reduced. Sample observation andillumination is also difficult to implement as described previously.

A further known method involving the use of a Cassegrain mirror systemis shown in FIG. 9 and is described in detail in U.S. Pat. No.5,117,108. This method has the merit of being free from chromaticaberrations, has a high spatial resolution and a near normal incidentangle but suffers the demerits of complexity and losses of power due tothe geometrical configuration. In this case, a laser beam 3 irradiatesthe first mirror 5 which contains an aperture 8. Light not reflectedcontinues on to irradiate the sample. The reflected light strikes thesecond mirror 1 which reflects and focuses the light onto the sample 2.Ions 4 generated by the irradiating light are accelerated along the axisof extraction 9 away from the sample 2 by an electric field and focusedthrough the aperture 8 by an electrostatic lens 7. The main merit ofthis system is the coincidence of the axes of extraction andirradiation. Primarily such systems are used in Time-of-Flight surfaceanalysis systems where very high laser powers are used. It, too, suffersthe demerit of the loss of laser power through the aperture as in theprevious example, FIG. 6, with the additional demerit that unfocusedlaser light 6 passes on to irradiate the sample but has the advantage ofbeing symmetrical about the extraction axis thereby simplifying theextraction optics and optimising the extraction efficiency. A furtherdemerit is the complexity and the cost of the system and the difficultyin implementing sample observation and illumination.

FIG. 10 shows a sample irradiated from the reverse side, and this isdescribed in U.S. Pat. No. 4,204,117. This has the advantage that thelaser probe is orthogonal and fully controllable from outside theevacuated chamber, but it is reliant on thin and very flat samples beingprepared on a substrate that is optically transparent at the wavelengthof the laser probe. In this example, laser beam 5 is focused by lens 3onto a sample 2. Ions 4 are emitted from the opposite side of the sample2 which is situated in an evacuated chamber, and these Ions acceleratedby an electric field through the grid 1. The main merit of this methodis the orthogonal irradiation of the sample from atmosphere wherecontrol is easy to implement, and the efficient extraction of ions alongthe extraction axis. The main demerits are the requirement for specialsample preparation and the difficulty of relating sample observation tothe sample irradiation position.

SUMMARY OF THE INVENTION

According to the invention there is provided an apparatus for theproduction and extraction of charged particles comprising a samplesubstrate upon which a sample is deposited, an optical element having atleast one reflective surface and having at least one hole extendingthrough the optical element, irradiation means for directing radiationonto a surface of the sample via said at least one reflective surface tostimulate emission of charged particles and extraction means forextracting at least some of the charged particles and directing theextracted charged particles away from said surface along an extractionaxis so that said charged particles pass from said sample through saidhole in the optical element, wherein the optical element has at leastone side surface inclined towards the sample, the or each side surfaceis disposed downstream of an opening to said at least one hole withrespect to the direction of extraction of the charged particles, andsaid at least one reflective surface is provided on a said side surface.

One advantage of this apparatus is that the axis of irradiation and theaxis of extraction are nearly coincident, therefore optimising theefficiency of the extraction and irradiation systems as previouslydiscussed. This invention satisfies both requirements without the needfor compromise. It also has the additional advantage that in the case ofa multi-faceted optical element multiple irradiation sources, samplevisualisation, illumination and scanning of the irradiating lightsource(s) can easily be incorporated using the same low cost opticalelement. Furthermore, different optical properties can be incorporatedon different facets of the element independently of the other facetswithout significantly altering the extraction efficiency. Cost ofmanufacture is relatively low and all other components in the opticalpath can be standard parts giving increased flexibility withoutintroducing additional cost. The electrostatic design is simplifiedsince the optical element can be manufactured from or coated byelectrically conductive material and preferably is symmetrical about theextraction axis in the critical areas close to the axis and cantherefore be designed to form an integral part of the extraction system.

The optical element itself can be a cone, pyramid or a similarly shapedsolid having a multi-sided base and sloping sides which project to meetat an apex, and the element may be truncated. The optical element has ahole or holes passing through it whose centre line or lines may beconcentric with or parallel to a line joining the geometric centre ofthe base to the projected apex.

In a preferred embodiment of the invention, said at least one reflectivesurface is inclined at an angle at or around 45° to the extraction axis.At least one said reflective surface may include a coating giving thesurface a specific reflection coefficient, and each of at least two ofsaid reflective surfaces may have a different coating giving a differentspecific reflection coefficient.

The at least one reflective surface may be flat or concave, and theoptical element may have a further surface which lies in a planeperpendicular to the extraction axis and faces the sample, and which maybe flat, concave, convex or a combination thereof. Said at least onehole may be circular, elliptical or of regular shape comprising two ormore curved segments or three or more straight segments. Alternatively,the hole may be of irregular shape comprising two or more curvedsegments or three or more straight segments.

The hole or holes may be covered by a mesh or grid.

BRIEF DESCRIPTION OF THE DRAWINGS

An embodiment of the invention will now be described, by way of exampleonly, with reference to the accompanying drawings of which:

FIG. 1 shows the principle of laser ablation with particular referenceto MALDI;

FIG. 2 shows typical angles and distribution of particles emitted fromthe surface of a sample during laser ablation;

FIGS. 3a and 3 b show the effects of shadowing of areas of the samplefrom the incident light irradiation due to surface roughness at an angleapproximately 45° to the axis of extraction;

FIGS. 4a and 4 b show the effects of shadowing of areas of the samplefrom the incident light irradiation due to surface roughness at anglesnearly coincident with the axis of extraction;

FIG. 5 shows a typical apparatus commonly used in a MALDI ToF massspectrometer;

FIG. 6 shows the use of a fibre optic wave guide used to direct a laserpulse to a sample;

FIG. 7 shows a common implementation of sample irradiation combined withsample observation;

FIG. 8 shows the use of a 45° mirror lying on the axis of extractionused to direct a laser beam onto the sample with a near perpendicularangle of incidence;

FIG. 9 shows the principle of the Cassegrain mirror used forperpendicular laser irradiation;

FIG. 10 shows a known method of ion generation stimulated by irradiationthrough the sample;

FIG. 11a shows schematically the configuration of a MALDI systemincorporating a preferred embodiment of the invention;

FIG. 11b shows schematically the optical element used in the system ofFIG. 11a, and

FIG. 12 shows the results of a computer simulation in the region aroundthe aperture of the optical element of FIG. 11b.

DESCRIPTION OF PREFERRED EMBODIMENT

In a preferred embodiment shown in FIG. 11b the optical elementcomprises a truncated four-sided pyramid 4 having an hole 7 in thecentre of the truncated face 3 passing through into a cavity 12 insidethe element. Each of the sloping sides comprises a reflective surface 1a,1 b,1 c,1 d.

Referring to FIG. 11a, the source of irradiation 18 typically, but notexclusively, a pulsed laser beam 16, is passed through a neutral densityfilter 17 which attenuates the power and is then reflected by means of amirror 15 to a focusing lens 5 and into an evacuated chamber 14 througha window 6. The laser beam 16 is reflected at the reflective surface 1 aof the optical element 4 and directed towards the sample 8 at a smallangle with respect to the extraction axis 2. In the case of thispreferred embodiment, this angle is approximately 4.5°. Chargedparticles generated by the laser beam are accelerated along the axis ofextraction 2 by electric fields supplied by the extraction elements 9.An electrostatic lens 10 focuses the extracted ions (indicated by thetrajectory envelope 19) at or near to the hole 7 in the pyramidaloptical element 4. The mirror support 11 and mirror 4 are held at thesame potential and form a part of the charged particle optical system.The charged particles are transmitted through the aperture into thecavity 12 and then into an optional electrostatic lens 13 for additionalfocusing. FIG. 12 shows a computer simulation of the region directly infront of the hole 3 in the front surface 4 of the pyramidal opticalelement 2. The lines of equal potential 1 are spaced at 10 voltseparation and show the minimal effects of the optical element 2 in theelectrostatic field. The cavity is kept nearly field-free in thisexample.

Sample visualisation is achieved by using a second reflective surface ofthe optical element to view the sample by means of a microscope systemexternal to the vacuum system. Such a system is commonly available andis not shown in detail here. Sample illumination can be implementedusing a third face of the optical element.

Additional light irradiation sources can be introduced to the sample byusing the fourth reflective surface of the optical element. Thisirradiation source can be employed simultaneously or sequentially.

A further advantage of this invention is that it is now possible to movethe laser across the reflective surface of the optical element therebyallowing scanning of an area of the sample. The areal power density iskept constant during the scanning due to the near perpendicular angle.This scanning technique is commonly used for imaging samples so thepossibility of obtaining chemical maps of a sample are now easily andeffectively achieved at low cost. Variable focusing of the laser spot isalso readily achieved with little or no degradation of the opticalperformance of the system.

What is claimed is:
 1. An apparatus for the production and extraction ofcharged particles comprising a sample substrate upon which a sample isdeposited, an optical element having at least one reflective surface andhaving at least one hole extending through the optical element,irradiation means for directing radiation onto a surface of the samplevia said at least one reflective surface to stimulate emission ofcharged particles and extraction means for extracting at least some ofthe charged particles and directing the extracted charged particles awayfrom said surface along an extraction axis so that said chargedparticles pass from said sample through said hole in the opticalelement, wherein the optical element has at least one side surfaceinclined towards the sample, the or each side surface is disposeddownstream of an opening to said at least one hole with respect to thedirection of extraction of the charged particles, and said at least onereflective surface is provided on a said side surface.
 2. An apparatusas claimed in claim 1, wherein the optical element has more than onesaid side surface disposed symmetrically about the extraction axis. 3.An apparatus as claimed in claim 1, wherein said optical element has afurther surface which lies in a plane perpendicular to the extractionaxis and faces said sample.
 4. An apparatus as claimed in claim 3wherein said further surface facing said sample is a flat surface.
 5. Anapparatus as claimed in claim 3, wherein said further surface facingsaid sample is a concave surface.
 6. An apparatus as claimed in claim 3wherein said further surface facing said sample is a convex surface. 7.An apparatus as claimed in claim 3, wherein said further surface facingsaid sample is a complex surface comprising flat, concave or convexcomponents.
 8. An apparatus as claimed in claim 1, wherein the opticalelement has more than one said side surface and said at least onereflective surface is provided on each of said side surfaces.
 9. Anapparatus as claimed in claim 1, wherein said optical element has theshape of a truncated pyramid and said at least one reflective surface isprovided on all or part of at least one angled side of the truncatedpyramid.
 10. An apparatus as claimed in claim 1, wherein said opticalelement has the shape of a truncated cone, said at least one reflectivesurface being provided on the conical surface of the truncated cone. 11.An apparatus as claimed in claim 1 in which said at least one reflectivesurface is inclined at or around an angle of 45° to said extractionaxis.
 12. An apparatus as claimed in claim 1 further comprising a meansof observation of said sample using at least one of said reflectivesurfaces.
 13. An apparatus as claimed in claim 1 further comprising ameans of illumination of said sample using at least one of saidreflective surfaces.
 14. An apparatus as claimed in claim 1 where thesaid optical element is made from or coated by a n electricallyconductive material.
 15. An apparatus as claimed in claim 1 where atleast one of said reflective surfaces has a coating to provide aspecific reflection coefficient.
 16. An apparatus as claimed in claim 15where each of at least two of said reflective surfaces has a differentsaid coating.
 17. An apparatus as claimed in claim 1 where the or eachsaid reflective surface is flat or concave.
 18. An apparatus as claimedin claim 1, wherein said charged particles are focused at or close tosaid hole in order to pass efficiently through it.
 19. An apparatus asclaimed in claim 1, wherein said irradiation means further includesmeans for scanning said radiation over said sample.
 20. An apparatus asclaimed in claim 1 wherein said irradiation means includes multiplesources producing radiation which is reflect at one or more of saidreflective surfaces.
 21. An apparatus as claimed in claim 20, whereinthe radiation derived from said multiple sources is applied eithersimultaneously or sequentially.
 22. An apparatus as claimed in claim 1,wherein said hole in said element is circular.
 23. An apparatus asclaimed in claim 1, wherein said hole in said element is elliptical. 24.An apparatus as claimed in claim 1, wherein said hole in said element isof regular shape comprising two or more curved segments.
 25. Anapparatus as claimed in claim 1, wherein said hole in said element is ofirregular shape, comprising two or more curved segments.
 26. Anapparatus as claimed in claim 1, wherein said hole in said element is ofregular shape, comprising three or more straight segments.
 27. Anapparatus as claimed in claim 1, wherein said hole in said element is ofirregular shape, comprising three or more straight segments.
 28. Anapparatus as claimed in claim 1 containing more than one of said holesin said element.
 29. An apparatus as claimed in claim 1, wherein saidhole or holes in said element are covered by a mesh or grid.